1. Field of the Invention
The present invention relates to the fields of ocular biology and treatments. More specifically, the present invention relates to, inter alia, methods for treating corneal disorders.
2. Description of the Related Art
Blindness from corneal etiologies is a serious global issue limiting the productivity and quality of life of approximately 4.9 million people around the world (1). The cornea is an avascular, translucent tissue that serves to allow the entry of light into the visual system and to focus the incoming rays of the visible spectrum. The outermost layer of the cornea, the stratified squamous epithelium, is integral in maintaining optical clarity and defending against microbial infection. The air-tear interface is the most important refracting surface in the ocular visual apparatus, and an irregular corneal epithelial surface results in substantial degradation of optical clarity, as is seen clinically with corneal abrasions, diabetic epitheliopathy, recurrent corneal erosions, dry eye syndrome, neurotrophic keratopathy, Stevens Johnson syndrome, ocular cicatricial pemphigoid, exposure keratopathy, and corneal transplantation, among others. Each of these corneal disease processes significantly compromises vision and consumes considerable resources in the United States in terms of work productivity, medical and pharmaceutical costs and quality of life.
The barrier function of the corneal epithelium serves a crucial role in maintaining ocular health by preventing microbial infection. Following trauma and chemical injuries of the corneal epithelium, the eye is markedly susceptible to infectious keratitis. Following surgical violation of the corneal epithelium, as occurs in cataract surgery, corneal transplantation surgery and refractive surgery [i.e., laser in situ keratomileusis (LASIK) and photorefractive keratectomy (PRK)], the risk of keratitis increases significantly. For example, keratitis after LASIK surgery was recently reported to occur in 2.66% of operated eyes (2). A tool to stimulate corneal epithelial healing would have a significant impact on visual morbidity from common ocular ailments, would improve visual rehabilitation after trauma and chemical injuries and would make surgical manipulations of the cornea safer and more reliable.
Keratinocytes form the epithelium of the skin, the epidermis. These cells undergo a distinct pattern of differentiation that is essential for the function of the skin as a protective barrier. This pattern is characterized by growth arrest and expression of the mature keratins 1 and 10 in the first differentiated layer of the epidermis, the spinous layer. Early differentiation in the spinous layer is followed by late differentiation in the granular layer accompanied by expression of proteins that are essential for the formation of the cornified envelope and corneocytes. The corneocytes constitute the outer layer of the epidermis, the stratum corneum, and give skin its resistance to mechanical stress (3). This program of keratinocyte differentiation can be regulated in vitro by the extracellular calcium concentration, with low calcium concentrations (<90 μM) promoting a proliferative phenotype and elevated calcium concentrations (>100 μM) stimulating differentiation (4, 5). The effects of extracellular calcium levels are thought to be physiologically relevant, since a calcium gradient (low in the basal layer and progressively higher in suprabasal layers) has also been observed in the epidermis in situ (6-9). Although the mechanism responsible for generation of this calcium gradient is unknown, presumably the extracellular calcium concentration regulates keratinocyte differentiation via activation of the G protein-coupled calcium-sensing receptor (CaSR) expressed in these cells (10-12).
Corneal epithelial cells exhibit many similarities to epidermal keratinocytes. Both cells form stratified epithelia exposed to the environment and express many of the same genes/proteins [e.g., the immature keratin 14 (13), the differentiation markers, involucrin, loricrin and transglutaminase (14, 15) and aquaporin-3 (16, 17)]. Elevated extracellular calcium concentrations inhibit the proliferation of both cell types (18), which can be grown in vitro in the same culture medium (15). Both cell types exhibit a programmed pattern of differentiation, including expression of mature keratins (keratins 1 and 10 in the epidermis and keratins 3 and 12 in the cornea) (18, 19). In addition, both cell types exhibit a migratory phenotype that is induced by epithelial wounding (20). Thus, it seems likely that the function of these cells is regulated by similar mechanisms.
Phospholipase D (PLD) hydrolyzes phospholipids, primarily phosphatidylcholine, to generate phosphatidic acid, which can be dephosphorylated by lipid phosphate phosphatases to yield diacylglycerol. Indeed, in several cell systems, phospholipase D activity has been shown to underlie at least a portion of agonist-induced sustained diacylglycerol production (21, 22). Diacylglycerol, in turn, is known to function as a second messenger (23), as is phosphatidic acid itself (24-30) and (31, 32). However, of interest is the fact that phospholipase D can also, in the presence of primary alcohols, catalyze a transphosphatidylation reaction to generate a phosphatidylalcohol. In fact, phospholipase D utilizes alcohols such as ethanol and butanol to yield phosphatidylethanol or -butanol (33), even when these alcohols are at low concentration.
In keratinocytes, one isoform of phospholipase D, phospholipase D2 (PLD2) is co-localized with the glycerol channel aquaporin-3 (AQP3) (34), and this isoform may be responsible for the observation that glycerol can be utilized by a phospholipase D enzymatic activity to generate in keratinocytes a potentially novel signaling lipid, phosphatidylglycerol (PG) (35, 36). Thus, AQP3 may provide glycerol to phospholipase D2 for the production of phosphatidylglycerol via the transphosphatidylation reaction, and this phosphatidylglycerol acts as a novel lipid signaling molecule to regulate early keratinocyte differentiation (36), as well as corneal epithelial cell function. Manipulations that alter the function of this PLD2/AQP3/PG signaling module can inhibit epidermal keratinocyte proliferation. Thus, stimulating phosphatidylglycerol formation by increasing AQP3 expression decreases the promoter activity of a marker of the proliferative basal layer (keratin 5) and enhances the promoter activity of a marker of differentiation (keratin 10) (35). Similarly, increasing phosphatidylglycerol production by raising the extracellular glycerol concentration also reduces keratinocyte proliferation, and direct provision of phosphatidylglycerol in the form of liposomes also inhibits the growth of rapidly proliferating keratinocytes (35). Interestingly, application of phosphatidylglycerol liposomes to slowly growing cells increases proliferation (35), suggesting that phosphatidylglycerol liposomes normalize keratinocyte function, accelerating growth in slowly proliferating cells and decreasing proliferation in rapidly growing cells. The data suggest that this signaling module also functions in corneal epithelial cells to produce phosphatidylglycerol (PG) and alter cell function.
How might phosphatidylglycerol act to alter keratinocyte and/or corneal epithelial cell function? One enzyme regulated by phosphatidylglycerol is PKC-_II; thus, in human leukemia cells PKC-βII is activated by nuclear phosphatidylglycerol, and this activation is required for cell cycle progression (47, 48). PKC-θ is also reportedly phosphatidylglycerol-activated (49) and mediates phosphorylation of the actin-binding domain of moesin. Another possible phosphatidylglycerol responsive protein kinase is “PK-P”, which has been isolated from human spleen (50-52). Alternatively, it is possible that phosphatidylglycerol can be reincorporated into the local membrane microdomains (lipid rafts) to regulate the organization of signaling molecules, such as the EGF receptor. Consistent with this idea, phosphatidic acid and phosphoinositide 3-kinase products have been reported to be concentrated in lipid rafts when platelets are stimulated by thrombin (53). Phosphatidylglycerol may also function by facilitating the interaction and function of membrane proteins, as has been observed in thylakoid membranes of spinach and a cyanobacterium, for which photosystem assembly requires phosphatidylglycerol (54, 55). Finally, phosphatidylglycerol and diphosphatidylglycerol (more commonly known as cardiolipin) are known to be important lipids in mitochondria. For instance, phosphatidylglycerol and cardiolipin restore the mitochondrial membrane potential in depleted mitochondria (56).
Thus, there is a continued need in the art for identification of compositions and methods to treat corneal disorders. The present invention fulfills this long-standing need and desire in the art.